植物抗软腐病基因工程的策略及研究进展

通过基因工程的手段提高植物对软腐病的抗性已被认为是一条切实可行的途径。目前已有大量研究证实,利用植物与病原菌互作的相关基因、病原菌致病性的相关基因以及杀菌肽基因可以或有望提高植物对软腐病的抗性。文章对以上几个方面及相关的抗软腐病基因工程的一些策略与研究进展进行了论述。     

中链脂肪酸抗菌和诱导防御肽表达的功能及其在仔猪饲料中的应用

近年,越来越多的研究表明,中链脂肪酸（MCFAs）抵抗病原菌是哺乳动物先天防御系统的重要组成部分,而且MCFAs也能够诱导人、猪和鸡内源性防御肽的表达,MCFAs的这些新功能尚未引起重视。MCFAs还和饲用有机酸或饲用植物精油具有协同抗菌增效作用,可以减少这些活性物质的使用量。相比于长链脂肪酸,日粮中添加MCFAs能显著地提高动物机体内氧气消耗量和线粒体呼吸速率,但产生活性氧自由基少,特别适合幼龄动物肠代谢和肝代谢所需的快速能量供应特点。日粮添加低浓度MCFAs（0.1%—0.5%,质量比）能显著提高新生或断奶仔猪存活率、粗蛋白粗脂肪消化率、饲料转化率,调节肠道菌群和改善肠上皮结构,进而促进生长。基于MCFAs的上述功能,将MCFAs与饲用有机酸或植物精油复配制备成微囊颗粒,可能是其用于仔猪抗生素替代品的尚佳方式。     

1株链霉菌BCG36的鉴定及抗菌活性研究

对分离自广西北海红树林根际土壤中的l株放线菌BCG36进行抗菌活性及生理生化特性的研究,并对其进行16S rDNA序列分析.结果显示:放线菌BCG36革兰氏染色呈阳性;有淀粉酶、纤维素酶、脂肪酶活性;牛奶先凝固后冻化,可使硝酸盐还原,不产硫化氢,不产黑色素,不使明胶液化;对大肠埃希菌、普通变形杆菌、乙型溶血性链球菌、金黄色葡萄球菌和白假丝酵母菌均有较好的抑制作用.16SrDNA测序后比对显示,放线菌BCG36菌株属于链霉菌属(Streptomyces).     

The Immune System of Marine Organisms as Source for Drugs against Infectious Diseases

The high proliferation of microorganisms in aquatic environments has allowed their coevolution for billions of years with other living beings that also inhabit these niches. Among the different existing types of interaction, the eternal competition for supremacy between the susceptible species and their pathogens has selected, as part of the effector division of the immune system of the former ones, a vast and varied arsenal of efficient antimicrobial molecules, which is highly amplified by the broad biodiversity radiated, above any others, at the marine habitats. At present, the great recent scientific and technological advances already allow the massive discovery and exploitation of these defense compounds for therapeutic purposes against infectious diseases of our interest. Among them, antimicrobial peptides and antimicrobial metabolites stand out because of the wide dimensions of their structural diversities, mechanisms of action, and target pathogen ranges. This revision work contextualizes the research in this field and serves as a presentation and scope identification of the Special Issue from Marine Drugs journal “The Immune System of Marine Organisms as Source for Drugs against Infectious Diseases”.     

抗菌肽的作用机理及其在养殖中的应用进展

抗菌肽是生物体内诱导产生的一类具有抗菌作用的生物活性肽,具有广谱的抗菌活性以及其他重要的生物学功能,被用作饲料添加剂广泛应用于畜禽生产中。文章综述了抗菌肽的理化特性、作用机理、生物学功能及其在养殖中的应用现状。     

Expression of Anti-Lipopolysaccharide Factor Isoform 3 in Chlamydomonas reinhardtii Showing High Antimicrobial Activity

Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene (ALFPm3), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii, which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 μg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii-derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 μM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon, providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.     

规模化蛋鸡场病原菌溯源与生物安全防控研究

细菌病严重威胁蛋鸡健康,以抗生素为主导的细菌病传统防控方式会导致病原菌耐药性严重、鸡蛋药物残留,影响蛋品安全。要保证蛋鸡产蛋期不用抗菌药物而又能生产出无菌、无抗的鸡蛋,是国内外技术难题。本团队以规模化蛋鸡场全封闭鸡舍为研究对象,基于PFGE、MLST、全基因组测序等技术,探明了蛋鸡病原菌的种类及基因组特征;对病原菌的耐药性研究表明,蛋鸡病原菌多重耐药严重,依靠药物防控的方式亟待转变;对规模化蛋鸡场细菌溯源研究结果表明,鸡舍生物媒介(鼠、苍蝇等)和非生物媒介(空气、饮水、饲料、鸡粪等)是蛋鸡病原菌的重要传染来源和传播途径;对规模化蛋鸡场生物安全量化评价结果表明,外部和内部生物安全权重高达92.2%。根据溯源和生物安全权重分析结果,提出了在规模化全封闭蛋鸡舍将蛋鸡细菌病从药物防控转变为生物安全防控的新策略;创新了规模化蛋鸡场细菌病防控的系统技术,实现蛋鸡规模养殖整个产蛋期不用抗生素,为生产无菌、无抗安全鸡蛋提供技术支撑。     

奶牛乳房炎抗性基因TAP cDNA克隆及序列分析

采用RT-PCR方法从奶牛乳腺组织中扩增气管抗菌肽(TAP)基因,重组到pMD19-T Simple载体中,并进行序列分析。序列分析结果显示,克隆的TAP基因包含完整的开放阅读框(ORF) 195 bp,与牛TAP基因同源性达93.8%;该ORF编码的64个氨基酸,含有β-防御素特征性结构即6个在特定位置上的保守半胱氨酸残基。TAP基因cDNA完整开放阅读框的克隆,为进一步开发应用重组牛β-防御素奠定了基础。     

噬菌体肽库技术筛选抗PRRSV肽及其应用

噬菌体展示肽库是一种被广泛用于抗原表位鉴定,结合蛋白筛选的技术。该试验利用噬菌体展示技术筛选与猪呼吸与繁殖障碍综合征病毒（PRRSV）ORF1B复制酶蛋白相互作用的蛋白,并进一步验证筛选蛋白的抗病毒作用。以表达纯化的PRRSV ORF1B蛋白CTD包被高亲和性96孔板作为靶蛋白,应用T7噬菌体展示技术对随机12肽库cDNA文库进行筛选,并分析测序筛选的克隆。将筛选出的克隆序列合成后,在体外验证合成多肽的抗PRRSV效果。结果表明,在4轮的噬菌体筛选后,共得到87个阳性克隆,经测序鉴定出11个筛选的12肽,并通过体外抗病毒试验得到P10是一个具有高抗病毒活性的12肽。试验结果为PRRSV的抗病毒研究奠定了基础。     

牛蛙(Rana catesbeiana)蛙皮抗菌肽基因的克隆、测序及其表达

利用RT—PCR的方法从牛蛙皮肤组织中克隆到大小为270bp的片段RCABP，将其克隆到pGEM—T载体，测序获得1个新的碱基序列。将此基因克隆到原核表达载体pQE一80L，获得融合表达质粒pQE一80L／DHFR／ABP，在1％IPTG诱导下进行表达。SDS—PAGE检测表明，重组蛙皮抗菌肽蛋白的表达量占菌体总蛋白的24％，以包涵体形式存在。体外抑菌试验表明，所构建的质粒能在大肠杆菌中表达具有体外抑菌活性的蛙皮抗菌肽，该融合蛋白具有良好的应用前景。